Secreted frizzled related protein fragments

ABSTRACT

The invention stems from the discovery that sFRP and fragments thereof can bind to members of the Wnt family of proteins and cause an increase in Wnt biological activity. Furthermore, fragments of sFRP that do not contain the CRD domain are shown to bind to Wnt proteins and modulate Wnt biological activity. Accordingly, the invention provides these sFRP fragments and variants of these fragments, as well as vectors and host cells containing nucleic acid sequences encoding the sFRP fragments and variants.

RELATED APPLICATIONS

This is a divisional of U.S. application Ser. No. 09/546,043, filed onApr. 10, 2000, now U.S. Pat. No. 6,600,018, issued Jul. 29, 2003, whichis incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to use of sFRP fragments and variants thereof tobind to members of the Wnt family of proteins and regulate Wntbiological activity.

BACKGROUND

Wnt proteins comprise a large family of structurally related,extracellular agents that have a variety of important functions duringembryonic development (Cadigan and Nusse, Genes Dev. 11:3286–3305, 1997and Dale, T. C., Biochem J. 329:209–223, 1998). They specify cellpolarity and fate, stimulate proliferation, and contribute to thepatterning of tissue in many animal models. Wnt signaling also has beenstrongly implicated in the development of neoplasia.

A set of secreted Fz-related proteins (sFRP or FRP) recently have beendescribed (Leyns et al., Cell 88:747–756, 1997; Wang et al., Cell88:757–766,1997; Rattner et al., Proc. Natl. Acad. Sci. U.S.A.94:2859–2863, 1997; Finch et al., Proc. Natl. Acad. Sci. U.S.A.94:6770–6775, 1997; Salic et al., Development 124:4739–4748, 1997;Melkonyan et al., Proc. Natl. Acad. Sci. U.S.A. 94:13636–13641, 1997;Pfeffer et al., Intl. J. Dev. Biol. 41:449–458, 1997; Mayr et al., Mech.Dev. 63:109–125,1997; Wolf et al., FEBS Lett. 417:385–389, 1997; Xu etal., Development 125:4767–4776, 1998; Chang et al., Hum. Mol. Genet.8:575–583, 1999; and Abu-Jawdeh et al., Lab. Invest. 79:439–447, 1999).These proteins consist ofapproximately 300 amino acids. including a CRD(cysteine rich domain) that is typically 30–50% identical to the CRDs ofFz family members. The carboxyl-terminal portion of these proteins oftencontains segments rich in positively charged residues, and two (sFRP-1and FrzB/sFRP-3) were reported to bind tightly to heparin (Finch et al.Proc. Natl. Acad Sci. U.S.A. 94:6770–6775, 1997 and Hoang et al. J.Biol. Chem. 271:26131–26137, 1996). The CRD has been also found to bethe Wnt binding site based on several experiments in which the Fz CRDconferred Wnt binding and/or responsiveness (Hsieh et al., Proc. Natl.Acad. Sci. U.S.A. 96:3546–3551, 1999; Bhanot et al., Nature 382:225–230,1996; and He et al., Science 275:1652–1654, 1997).

SUMMARY

The invention stems from the discovery that sFRP and fragments thereofcan bind to members of the Wnt family of proteins, and furthermore thatthese molecules have a biphasic effect on Wnt activity. At highconcentrations these proteins inhibit Wnt activity and at lowconcentrations these proteins increase in Wnt biological activity.Furthermore, fragments of sFRP that do not contain the CRD domain areprovided and these fragments are shown to bind to Wnt proteins andmodulate Wnt biological activity.

Accordingly, the invention provides fragments of sFRP which are able tobind to Wnt thereby modulating Wnt biological activity. These sFRPfragments may (SEQ ID NOS: 5–7) or may not (SEQ ID NO: 8) contain theCRD of sFRP. Because these fragments bind to Wnt these fragments, andvariants thereof, can be used to screen for other molecules that bind toWnt and modulate Wnt activity.

The invention also provides methods of using sFRP-1 and fragmentsthereof to increase Wnt biological activity. The increase in Wntactivity is desirable for treating developmental disorders that areassociated with decreased Wnt biological activity as well as forinducing the development of neoplasias which is desirable inexperimental models for the study of tumor growth.

The invention also provides methods of using sFRP without the CRD domainto increase or decrease Wnt biological activity depending upon theamount provided. Such methods are useful for treating disordersassociated with increased Wnt biological activity and for thesuppression of tumor growth. Furthermore, the finding that sFRPfragments without the CRD domain bind to Wnt proteins allows for thedevelopment of screening assays which identify small molecules or othercompounds which may block sFRP/Wnt binding or enhance sFRP/Wnt binding.Thus, for example, the invention provides methods of identifying smallmolecules or binding proteins that bind either Wnt or fragments of sFRPwithout the CRD and disrupt sFRP/Wnt binding.

Accordingly, another aspect of the invention provides methods ofmodulating Wnt protein biological activity. These methods involvecontacting at least one Wnt protein with least one sFRP fragment orvariant thereof and producing an increase Wnt biological activity.

Yet another aspect of the invention provides a sFRP fragment that doesnot contain the CRD portion of sFRP (SEQ ID NO: 8), but yet maintainsWnt binding activity. Accordingly this fragment and variants thereof canbe used to screen for other molecules that bind to Wnt and modulate Wntbiological activity.

When used to modulate Wnt biological activity the fragments describedabove can be used to further characterize the biological role that Wntplays in the various developmental processes. Furthermore, thesefragments can also be used to modulate conditions associated withabnormal Wnt biological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes a graph and three gels that depict the results from thepurification of recombinant sFRP-1. FIG. 1A is a graph that shows theisolation of sFRP-1 from sFRP-1/MDCK cells (ATCC NO. CCL-34) as thesFRP1 was eluted from a heparin-Sepharose resin (bed volume, 1 ml).Samples were eluted with increasing NaCl concentration (dashed line),and protein content was assessed by measuring optical density at 280 nm(solid line). Fractions (1 ml) are indicated on the horizontal axis. Thethick bar indicates fractions containing sFRP-1. FIG. 1B is a 12%SDS-PAGE gel in which selected fractions from the heparin-Sepharoseresin (described in A) were separated in. The protein bands werevisualized by silver staining. The molar concentration of NaCl foreluted fractions is indicated above the lanes. The positions ofmolecular mass markers are shown at the left. The inset shows silverstaining of three 1.0 M NaCl fractions resolved in an 8% polyacrylamidegel. FIG. 1C is an anti-sFRP-1 immunoblot. The samples are from the samefractions viewed in B, again separated by 12% SDS-PAGE. FIG. 1D is ananti-sFRP-1 immunoblot of conditioned media from clonal lines derivedfrom sFRP-1/MDCK mass culture.

FIG. 2 includes a schematic diagram of the relationship between thevarious sFRP-1 derivatives, an immunoblot using the various sFRP-1derivatives, and a gel showing the elution profile of the various sFRP-1derivatives. These figures taken together identify the sFRP-1heparin-binding domain. FIG. 2A is a schematic of sFRP-1 and itsderivatives. Numbers indicate amino acid residues in sFRP-1 sequence atboundaries of recombinant proteins. CRD (hatched boxes) borders also areshown. The white boxes correspond to lysine-rich segments. M/H indicatesthe Myc-His epitope tags. FIG. 2B is an anti-Myc immunoblot (left panel)and silver stain (right panel) analysis of purified sFRP-1 mutantproteins. The positions of molecular mass markers are indicated at theleft. FIG. 2C is an immunoblot showing the elution pattern of the sFRP-1derivatives from a heparin-sepharose. The derivatives were isolated fromconditioned media from MDCK cells transfected with sFRP-1 derivativesand applied to heparin-Sepharose columns. Samples were eluted withindicated concentrations of NaCl, and fractions were analyzed by Westernblotting with anti-Myc.

FIG. 3 includes a gel and three graphs which show the results of anELISA demonstrating sFRP-1/Wg binding. FIG. 3A is a graph of the resultsfrom an ELISA. Wells were coated with sFRP-1 or BSA alone and incubatedwith dilutions of Wg-containing or S2 control medium. Bound Wg proteinwas detected with anti-Wg and secondary immune reagents as describedunder “Experimental Procedures.” FIG. 3B is a gel showing the Wgcross-reactive protein pattern from conditioned media from control S2 orWg-expressing S2 cells that were analyzed by immunoblotting withanti-Wg. The arrow at the right indicates primary Wg band. Positions ofmolecular mass markers are shown at the left. FIG. 1C is a graph showingthe results from an ELISA. Wells were coated with sFRP-1 derivatives andincubated with indicated dilutions of conditioned media containing Wg.FIG. 3D is a graph showing the results from another ELISA. Wells werecoated with sFRP-1 and incubated with Wg-containing media that had beenpre-incubated with the indicated concentrations of sFRP-1 derivatives.Each panel is representative of several experiments.

FIG. 4 is a gel showing the results of co-precipitation assays of sFRP-1derivatives and Wg. sFRP-1 mutant proteins were incubated withWg-containing media, precipitated with anti-Myc, and immunoblotted withanti-Wg (upper panel) or anti-Myc (lower panel). Serial dilutions of Wgmedium were also analyzed. Note that sFRP-Δ1 (SEQ ID NO: 5) migratednear the bottom of the gel in the lower panel. The positions ofmolecular mass markers are shown at the right. IP, immunoprecipitation.

FIG. 5 includes three gels showing the results from covalentcross-linking assays of sFRP-1 and Wg. FIG. A is a gel showing theresults from an ¹²⁵I-sFRP-1 incubation with medium from S2 orWg-expressing S2 cells, followed by addition of BS 3 cross-linkingagent. In some reactions, unlabeled sFRP-1 (1.7 mM) and/or heparin (10μg/ml) were also present. Proteins immunoprecipitated with anti-Wg wereseparated by 8% SDS-PAGE and processed for autoradiography. Smaller andlarger cross-linked complexes are indicated by arrowhead and arrow,respectively. The positions of molecular mass markers are shown at theleft. FIG. 5B is gel showing the results from a competition assay withunlabeled sFRP-1. FIG. 5C is another gel showing the effect of varyingheparin concentrations on cross-linking.

FIG. 6 includes four gels showing the results from Arm stabilizationassays that tested the biological activity of sFRP-1 and itsderivatives. FIG. 6A is a gel showing the results from DFz2-expressingS2 cells that were incubated with Wg medium at the indicatedconcentrations of sFRP-1. Cell lysates were analyzed by immunoblottingwith anti-Arm (upper panel) and anti-HSP70 (lower panel). Similarexperiments as in A were performed with sFRP-M/H (SEQ ID NO: 4; B),sFRP-ΔCRD (SEQ ID NO: 8; C), and sFRP-Δ2 (SEQ ID NO: 6; D). Each panelis representative of three to five separate experiments.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 shows the cDNA sequence of human sFRP-1.

SEQ ID NO: 2 shows the nucleic acid sequence of the human sFRP-1 openreading frame.

SEQ ID NO: 3 shows the amino acid sequence of human sFRP-1.

SEQ ID NO: 4 shows the amino acid sequence of human sFRP-1-M/H.

SEQ ID NO: 5 shows the amino acid sequence of human sFRP-Δ1.

SEQ ID NO: 6 shows the amino acid sequence of human sFRP-Δ2.

SEQ ID NO: 7 shows the amino acid sequence of human sFRP-Δ3.

SEQ ID NO: 8 shows the amino acid sequence of human sFRP-ΔCRD.

SEQ ID NO: 9 shows the nucleic acid sequence encoding sFRP-1-M/H.

SEQ ID NO: 10 shows the nucleic acid sequence encoding sFRP-Δ1.

SEQ ID NO: 11 shows the nucleic acid sequence encoding sFRP-Δ2.

SEQ ID NO: 12 shows the nucleic acid sequence encoding sFRP-Δ3.

SEQ ID NO: 13 shows the nucleic acid sequence encoding sFRP-ΔCRD.

DETAILED DESCRIPTION

I. Abbreviations

Arm, armadillo protein; CRD, cysteine-rich domain; sFRP, secretedFrizzled-related protein; MDCK, Madin-Darby canine kidney; BSA, bovineserum albumin; HSPG, heparin-sulfate proteoglycan; ELISA, enzyme-linkedimmunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PBS,phosphate-buffered saline; mAb, monoclonal antibody; BS 3, Wnt, Wntproteins; bis(sulfosuccinimidyl) suberate; M/H, Myc-His epitope tags.

II. Definitions

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

sFRP: sFRP is a secreted protein that consists if approximately 300amino acids, including a CRD that is typically between 30% and 50%identical to the CRDs of the Fz protein family members. There areseveral different sFRP proteins and the nucleic acid sequence of theprototypical member, sFRP-1, is provided in SEQ ID NO: 1. The nucleicacid and amino acid sequences of other members of the sFRP family can befound at the National Center for Biotechnology Website, for exampleunder the accession numbers, AF218056 (Gallus gallus FRP-2), AV354083(Mus musculus-FRP-1), AV304328 (Mus musculus s-FRP-2), U24163 (homosapiens sFRP-3/FrzB) and AI587049 (Homo sapiens sFRP-1). The openreading frame of the prototypical sFRP is shown in SEQ. ID NO: 2, whilethe sequence of the protein is shown in SEQ. ID NO: 3. The presentinvention takes advantage of the discovery that particular sFRP andfragments of sFRP can increase Wnt biological activity. Furthermore,this activity and binding to Wnt proteins generally is shown not to bedependent upon the CRD region of sFRP.

sFRP-1 binding activity and its ability to modulate Wnt biologicalactivity may be assayed by methods described herein. The ability of afragment of sFRP-1 protein to perform these activities is believed to bebeneficial in a number of applications, including clinical applicationssuch as tumor therapy and treatment of diseases with abnormal Wntactivity.

While the amino acid sequence of the prototypical sFRP is shown in SEQ.ID NO: 3, one of skill in the art will appreciate that variations inthis amino acid sequence, such as 1, 2, 5, or 10, deletions, additions,or substitutions, may be made without substantially affecting theactivities of the protein (or fragments of the protein) discussed above.Thus, the term “sFRP” fragments encompasses both the proteins having theamino acid sequences shown in SEQ. ID NOs: 4–8, as well as amino acidsequences that are based on these sequences but which include one ormore sequence variants. Such sequence variants may also be defined inthe degree of amino acid sequence identity that they share with theamino acid sequence shown in SEQ. ID NOs: 4–8. Typically, sFRP sequencevariants will share at least 80% sequence identity with the sequencesshown in SEQ. ID NOs: 4–8. More highly conserved variants will share atleast 90%, at least 95%, or at least 98% sequence identity with thesequences shown in SEQ. ID NOs: 4–8. In addition to sharing sequenceidentity with the prototypical sFRP protein sequence, such sequencevariants possess the ability to bind to Wnt proteins and/or modulate Wntbiological activity.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Polypeptide: A protein fragment including at least two amino acidresidues.

Polynucleotide: A nucleic acid sequence including at least two nucleicacid residues.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art. A vector may alsoinclude a sequence encoding for an amino acid motif that facilitates theisolation of the desired protein product such as a sequence encodingmaltose binding protein, c-myc, or GST.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Isolated: An “isolated” biological component (such as a nucleic acid orprotein or organelle) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of sFRP will possess a relatively high degree ofsequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237–244, 1988; Higginsand Sharp, CABIOS 5:151–153, 1989; Corpet et al., Nucleic Acids Research16:10881–10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119–129, 1994.

The NCBI Basic Local Alignment Search Tool (BLAST™) (Altschul et al., J.Mol. Biol. 215:403–410, 1990.) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

Variants of sFRP fragments are typically characterized by possession ofat least 50% sequence identity counted over the full length alignmentwith the amino acid sequence of the sFRP fragment using the NCBI Blast2.0, gapped blastp set to default parameters. For comparisons of aminoacid sequences of greater than about 30 amino acids, the Blast 2sequences function is employed using the default BLOSUM62 matrix set todefault parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the Blast 2 sequencesfunction, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). Proteins with even greater similarity tothe reference sequences will show increasing percentage identities whenassessed by this method, such as at least 60%, at least 65%, at least70%, at least 75%. at least 80%, at least 90% or at least 95% sequenceidentity. When less than the entire sequence is being compared forsequence identity, homologs and variants will typically possess at least75% sequence identity over short windows of 10–20 amino acids, and maypossess sequence identities of at least 85% or at least 90% or 95%depending on their similarity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe website that is maintained by the National Center for BiotechnologyInformation in Bethesda, Md. One of skill in the art will appreciatethat these sequence identity ranges are provided for guidance only; itis entirely possible that strongly significant homologs could beobtained that fall outside of the ranges provided.

CRD: A cysteine rich domain that typically is about 120 amino acids inlength and found on the amino terminal half of Fz proteins. In theprototypical sFRP described herein the CRD stretches from amino acidnumber 57 through 165.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

Mimetic: A molecule (such as an organic chemical compound) that mimicsthe activity of a protein, such as a sFRP fragment and variants thereof.Peptidomimetic and organomimetic embodiments are within the scope ofthis term, whereby the three-dimensional arrangement of the chemicalconstituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the peptide backbone and componentamino acid sidechains in the peptide, resulting in such peptido- andorganomimetics of the peptides having substantial specific inhibitoryactivity or agonist activity. For computer modeling applications, apharmacophore is an idealized, three-dimensional definition of thestructural requirements for biological activity. Peptido- andorganomimetics can be designed to fit each pharmacophore with currentcomputer modeling software (using computer assisted drug design orCADD). See Walters, “Computer-Assisted Modeling of Drugs”, in Klegerman& Groves, eds., Pharmaceutical Biotechnology, Interpharm Press: BuffaloGrove, Ill., pp. 165–174, 1993 and Principles of Pharmacology (ed.Munson), chapter 102, 1995, for a description of techniques used incomputer assisted drug design.

These and other aspects of the invention are explained in more detail inthe following sections. Additionally, throughout the specification andclaims, reference to the singular (such as “a” or “the”) includes theplural, unless clearly indicated otherwise by context.

III. Development of Methods of Controlling the Biological Activity ofWnt

A. Results

1. Recombinant Expression of sFRP-1 and its Derivatives for FunctionalStudies

To generate a plentiful supply of sFRP-1 protein, MDCK cells weretransfected with a pcDNA3.1 vector containing the coding sequence ofhuman sFRP-1 (SEQ ID NO: 2). MDCK cells have favorable properties forrecombinant expression because they grow rapidly and, once confluent,can remain attached to plastic for several weeks in serum-free medium.Consequently, several sequential harvests of conditioned medium can becollected from the same monolayer. A one-step preparative schemeinvolving heparin-Sepharose affinity chromatography was sufficient topurify sFRP-1 from concentrated conditioned medium (FIG. 1A). Typically0.25–0.50 mg of sFRP-1/liter of medium was recovered from thetransfected mass culture. Silver staining and immunoblot analysisconfirmed the purity and identity of the recombinant protein that elutedfrom heparin-Sepharose with 1.0 M NaCl (FIGS. 1, B and C). The proteinband in both analyses usually was broad and occasionally resolved intotwo or three components (FIG. 1B, inset), indicative ofmicroheterogeneity.

The individual components resolved in the blotting procedure describedabove were identified by microsequencing. The microsequencing revealedthat the majority of the protein had an amino-terminal sequencebeginning at Ser-31, one residue downstream from the proposed signalpeptide cleavage site (Finch et al., Proc. Natl. Acad. Sci. U.S.A.94:6770–6775, 1997). Two other sequences, beginning at Asp-41 andPhe-50, also were obtained and presumably resulted from partialproteolysis. Glycosylation may account for additional heterogeneity.

To optimize the yield of recombinant protein, clonal lines were isolatedfrom the mass culture, and their conditioned media were screened forsFRP-1 content (FIG. 1D). Clone 11 cells (lane 11 in FIG. 1D) wereexpanded for large scale preparations and yielded 2–4 mg of sFRP-1/literof conditioned medium.

The deletion mutants that were generated allowed for the correlation ofbinding properties with particular regions of the sFRP-1 molecule. Tofacilitate detection and purification of the deletion mutant c-Myc andpolyhistidine epitope tags were attached to the carboxyl terminus ofeach derivative (FIG. 2A). The sFRP-Δ1 (SEQ ID NO: 5) sequence extendsthrough amino acid residue 171, a short distance beyond the CRD. sFRP-Δ2(SEQ ID NO: 6) and sFRP-Δ3 (SEQ ID NO: 7) contain progressively more ofthe carboxyl-terminal region. Included within sFRP-Δ3 (SEQ ID NO: 7) isa lysine-rich domain previously identified as a consensus binding sitefor hyaluronic acid (Finch et al., Proc. Natl. Acad Sci. U.S.A.94:6770–6775, 1997). Finally, the sFRP-ΔCRD (SEQ ID NO: 8) deletionmutant was generated such that it lacks the CRD but contains theremaining amino-terminal and entire carboxyl-terminal sequences.

All the sFRP-1 derivatives were readily secreted and remained insolution after ultrafiltration, chromatography, dialysis, and repeatedfreeze-thawing, suggesting that there were no gross defects in folding.The proteins were purified to homogeneity by using nickel resinchromatography (FIG. 2B). Initial characterization of these moleculesfocused on their heparin-binding properties because of the potentialimportance of this binding trait to the interaction with Wnt proteins.Although full-length sFRP-1 labeled with the c-Myc and histidine tags(sFRP-M/H; SEQ ID NO: 4) eluted from heparin-Sepharose in the sameposition as native sFRP-1 , sFRP-Δ1 (SEQ ID NO: 5) and sFRP-Δ2 (SEQ IDNO: 6) were not retained on the resin (FIG. 2C). Inclusion of thelysine-rich segment in sFRP-Δ3 (SEQ ID NO: 7) resulted in a protein withintermediate heparin-binding capability, eluting with 0.5 M NaCl. Thisimplied that the heparin-binding properties of intact sFRP-1 probablyinvolve multiple sites distributed in the carboxyl-terminal third of themolecule. Consistent with this view, sFRP-ΔCRD (SEQ ID NO: 8) boundheparin-Sepharose in a manner similar to that of the native protein(FIG. 2C).

2. Binding Assays with Recombinant sFRP-1 and Derivatives, UsingWingless (Wg) as a WNT Prototype

Wingless (Wg) is a gene that was discovered in Drosophila melanogasterthat codes for a protein in the WNT family. The various assays describedbelow show that Wg binds to sFRP-1. First, an ELISA was used to measuresFRP-1 binding to Wg. Wells were coated with purified full-length sFRP-1and then blocked with an excess of BSA. Subsequently, conditioned mediumfrom S2HSWg cells expressing soluble Wg was incubated in the wellsovernight at room temperature. As a control, aliquots of the same mediumwere incubated in wells treated with BSA but not sFRP-1. In addition,other wells coated with sFRP-1 were incubated with medium from S2 cellsthat did not express Wg. As illustrated in FIG. 3A, Wg boundspecifically to the wells coated with sFRP-1, and the amount of bound Wgvaried with the dilution of Wg medium. In contrast, little Wg wasdetected in wells that had not been treated with sFRP-1, and no signalwas observed when medium lacking Wg was used in the assay (FIGS. 3, Aand B). These results indicate that sFRP-1 can bind Wg and presumablyother Wnt proteins as well.

Based on these findings, a similar analysis was performed using wellscoated with the various sFRP-1 deletion mutants (FIG. 3C). Surprisingly,the data indicated that the CRD was not required for Wg binding. Infact, the amount of Wg detected in wells coated with sFRP-ΔCRD (SEQ IDNO: 8) matched that seen in wells treated with full-length,epitope-tagged sFRP-1.

On the other hand, derivatives that contained the CRD domain and lackedportions of the carboxyl-terminal region showed reduced Wg binding.sFRP-Δ2 (SEQ ID NO: 6) exhibited intermediate binding activity, whereassFRP-Δ1 (SEQ ID NO: 5) and sFRP-Δ3 (SEQ ID NO: 7) had only limitedbinding activity. No binding was observed in wells treated with BSAalone (data not shown). These results indicate that thecarboxyl-terminal region of sFRP-1 was primarily responsible for itsability to bind Wg.

In the experiments described above, wells were coated in parallel withthe same molar concentration of the various sFRP-1 derivatives, andanalysis indicated that comparable amounts of each derivative adhered tothe well surface. Therefore, the contrasts in relative bindingefficiency were not attributable to differences in the concentration ofprotein coating the wells. Given the above described results, it wasconceivable that the sFRP-1 derivatives could adsorb to the well surfacein ways that would differentially mask a Wg binding site, however, thiswas shown not to be the case in subsequent binding assays performed insolution.

Wg binding in solution was tested by coating wells with native sFRP-1,Wg medium that had been preincubated for 45 minutes with varyingconcentrations of the sFRP-1 mutants was then added. The ability of themutants to interact with Wg was indicated by the extent to which theycould inhibit Wg binding to the wells. The results of these experiments(FIG. 3D) were in agreement with the previous pattern: sFRP-ΔCRD (SEQ IDNO: 8) competed for Wg binding as effectively as sFRP-M/H (SEQ ID NO:4), whereas sFRP-Δ2 (SEQ ID NO: 6) had a partial effect. sFRP-Δ1 (SEQ IDNO: 5) and sFRP-Δ3 (SEQ ID NO: 7) had little or no efficacy in thecompetition assay. Thus, the observed differences in Wg binding to thesFRP-1 derivatives were not caused by inadvertant masking of bindingsites but were due to the intrinsic properties of the derivatives.

The association of sFRP-1 proteins with Wg was also examined inco-precipitation experiments. Following incubation of epitope-taggedsFRP-1 mutants with Wg medium, proteins were precipitated with anti-Mycand subsequently immuno-blotted with anti-Wg (FIG. 4). Approximately10–20% of Wg protein was precipitated with either sFRP-ΔCRD (SEQ ID NO:8) or sFRP-M/H (SEQ ID NO: 4). A weak association was detected withsFRP-Δ2 (SEQ ID NO: 6), but none was observed with sFRP-Δ1 (SEQ ID NO:5) or sFRP-Δ3 (SEQ ID NO: 7). Thus, both ELISA and co-precipitationexperiments showed that the CRD was not required for Wg binding.

To rule out the possibility that an unidentified factor in the Wg mediummight be responsible for mediating the binding interaction betweensFRP-1 and Wg, covalent affinity cross-linking studies were performedwith radiolabeled sFRP-1 and conditioned medium from Wg-expressing andcontrol S2 cells. Following incubation of reactants as described under“Experimental Procedures,” proteins were immunoprecipitated with anti-Wgand resolved by SDS-PAGE, and cross-linked complexes were detected byautoradiography (FIG. 5A). No complexes were observed in the absence ofcross-linker or Wg. In contrast, two distinct radiolabeled bands wereevident when the cross-linking reaction was carried out in the presenceof Wg. The lower band had an apparent molecular mass consistent with acomplex comprised of one molecule each of sFRP-1 and Wg, therebyindicating that the two proteins can interact directly with each other.

The difference in apparent size of the upper and lower bands was 35±2.9kDa (mean±S.D., calculated from four experiments), which correspondsclosely to the molecular mass of sFRP-1 (Finch et al., Proc. Natl. Acad.Sci. U.S.A. 94:6770–6775, 1997). This suggested that the upper band is acomplex containing two sFRP-1 molecules and one Wg molecule. Anotherpossibility is that the upper band represents a ternary complex with athird unidentified partner linked to sFRP-1 and/or Wg. The absence ofboth bands when Wg was lacking from the cross-linking reaction, whenanti-Wg immunoprecipitation was omitted, or in the presence of an excessof unlabeled sFRP-1 demonstrated that sFRP-1 and Wg were present in bothcomplexes (FIG. 5A and data not shown). Comparable displacement of¹²⁵I-sFRP-1 by unlabeled sFRP-1 indicated that the binding affinity oftracer in the two complexes was similar (FIG. 5B). Unlabeled sFRP-ΔCRD(SEQ ID NO: 8) and sFRP-Δ2 (SEQ ID NO: 6) also competed with tracer forbinding in both complexes, although neither was as potent as full-lengthsFRP-1.

Because sFRP-1 and Wg are both heparin-binding proteins and becauseheparin-sulfate proteoglycan (HSPG) had been shown to regulate Wg/Wntactivity in vivo, the role of heparin was examined in theabove-described cross-linking experiment. Initial studies revealed thatheparin at a concentration of 10 μg/ml caused a dramatic increase in theintensity of both bands corresponding to cross-linked complexes (FIG.5A). Subsequently, a dose-response analysis indicated a biphasic patternin which optimal stimulation was observed with 1–10 μg/ml of heparin(FIG. 5C). This effect was specific for heparin, because no stimulationwas observed when chondroitin sulfate, keratin sulfate, or hyaluronicacid was used under similar conditions (data not shown). These dataindicated that heparin and presumably HSPG have a marked impact on theinteraction of sFRP-1 and Wnt proteins, as represented by Wg in thisstudy.

Subsequent assays showed that sFRP-1 has a biphasic effect onWg-dependent stabilization of Armadillo protein. These assays alsotested the biological activity of recombinant sFRP-1 derivatives.Because Wg had been used in the binding experiments, sFRP-1 activity wasexamined in a Wg-dependent bioassay. As previously reported (Bhanot etal., Nature 382:225–230, 1996), soluble Wg increases the steady-statelevel of Arm in cells engineered to express DFz2 (Drosophila frizzled 2)(FIG. 6A). Inhibition of Wg/DFz2 signaling by sFRP-1 was expected, givenearlier reports that sFRP-1 and other sFRP family members antagonizedWg-dependent and other Wnt-dependent duplication of the dorsal axis inearly Xenopus embryos (Leyns et al., Cell 88:747–756, 1997; Wang et al.,Cell 88:757–766, 1997; Rattner et al., Proc. Natl. Acad. Sci. U.S.A.94:2859–2863, 1997 and Xu et al., Development 125:4767–4776, 1998).Indeed, high concentrations of sFRP-1 (10 and 25 mg/ml) blocked Wgactivity (FIG. 6A). However, lower concentrations of sFRP-1 had theopposite effect: as little as 20 ng/ml (0.6 nM) of sFRP-1 incubated withWg medium caused a significant increase in the amount of Arm proteinrelative to that observed with Wg medium alone. Maximal Arm response wasseen with 100–500 ng/ml of sFRP-1. This potentiating effect was notattributable to a prolongation of the Wg half-life in solution, becauseWg half-life was much longer than the duration of the assay, even in theabsence of sFRP-1. sFRP-1 had no effect on Arm levels in the absence ofWg and no effect on S2 cells lacking DFz2 expression (data not shown).Thus, sFRP-1 activity presumably involved an interaction with Wg thatrequired signaling through DFz2.

The arm assay was also used to compare the effect of sFRP-M/H (SEQ IDNO: 4), sFRP-ΔCRD (SEQ ID NO: 8), and sFRP-Δ2 (SEQ ID NO: 6) on Wntbiological activity. sFRP-M/H (SEQ ID NO: 4) behaved like native sFRP-1at the concentrations tested (0.02–2 mg/ml), enhancing Wg-dependentstabilization of Arm (FIG. 6B). This implied that the addition of Mycand histidine epitope tags did not alter its biological activity.sFRP-ΔCRD (SEQ ID NO: 8) and sFRP-Δ2 also increased the activity of Wgin this assay, although their potency was reduced, especially that ofsFRP-Δ2 (SEQ ID NO: 6), relative to sFRP-M/H (FIG. 6, B–D). Takentogether, these results demonstrated that the recombinant proteins usedin the binding analysis were biologically active. This reinforced theconclusions drawn above concerning the structural requirements for Wgbinding. In particular, the CRD was not required either for binding orbiological activity, although its absence reduced the specific activityof sFRP-1.

B. Discussion

The present disclosure demonstrates that sFRP-1 and Wnt protein binddirectly to each other. Previous reports described co-precipitationexperiments in which various sFRP family members were shown to associatewith one or more Wnt proteins. These results were inconclusive because,they did not address the possibility that their association might beindirect, mediated by a factor that could bind both proteins. This was adistinct possibility because neither protein was used in a purifiedstate. In addition, some of the earlier observations were made withcells co-expressing both recombinant proteins such that associationmight occur during their synthesis and would not reflect a normalpattern of interaction. However, the studies described herein minimizedthe contribution of indirect effects by using purified preparations ofsFRP-1 and an independent source of Wg. The sFRP-1/Wg binding wasdemonstrated both in solid phase and solution assays, utilizing ELISAand co-precipitation formats. Covalent cross-linking of ¹²⁵I-sFRP-1 withWg provided strong evidence of a direct interaction between the twoproteins. Surprisingly, besides detecting a cross-linked complexconsistent in size with one sFRP-1 and one Wg molecule, a larger complexwhose size suggested the presence of a second sFRP-1 molecule was alsoobserved. Although the exact nature of this larger entity is currentlyunknown, taken together these results established for the first timethat sFRP-1 is a direct binding partner for Wnt protein.

Additionally, the ¹²⁵I-sFRP-1/Wg cross-linked complexes were detected inthe absence of added heparin but were more abundant when the reactionwas performed with an optimal concentration of exogenous heparin.Heparin or endogenous HSPG is believed to promote sFRP-1/Wg binding byserving as a scaffold to facilitate interaction between sFRP-1 and Wg.Alternatively, heparin/HSPG may promote binding by stabilizing aconformation of either sFRP-1 or Wg that would increase their mutualaffinity or by enhancing ligand or receptor oligomerization. However,the ability to bind heparin was not itself sufficient for cross-linkingto Wg; similar experiments conducted with Wg medium and a controlheparin-binding polypeptide did not yield cross-linked Wg complexes(data not shown). Moreover, the spacer arm of the cross-linking agentwas only 11.4 Å long, reinforcing the conclusion that sFRP-1 bindsdirectly to Wg and presumably other Wnt proteins. Although the effect ofheparin on sFRP-1 /Wg binding was observed in an artificial, cell-freesetting, these results are consistent with other findings suggesting animportant role for HSPG in Wnt signaling in vivo. The present findingsindicate that HSPG has a profound effect on Wnt activity andspecifically indicate that HSPG can regulate Wnt binding interactionswith sFRP proteins.

Among the most unexpected findings was the observation that the CRD wasnot required for Wg binding. The prevailing view that the CRD is the Wntbinding site is based on several experiments in which the Fz CRDconferred Wnt binding and/or responsiveness (Hsieh et al., Proc. Natl.Acad Sci. U.S.A. 96:3546–3551, 1999; Bhanot et al., Nature 382:225–230,1996; and He et al., Science 275:1652–1654, 1997).

Evidence that sFRP-ΔCRD (SEQ ID NO: 8) can bind Wg as shown above inmultiple experimental models and was highly reproducible. The proteinswere shown to interact both in a solid phase assay and in solution.sFRP-ΔCRD (SEQ ID NO: 8) also retained the full heparin-binding capacityof the native protein. Therefore, it is possible that this sFRP-1derivative associated with Wg via soluble HSPG, whose presence inWg-containing S2 conditioned medium had been previously inferred(Reichsman et al., J. Cell. Biol. 135:819–827, 1996). Such a complexwould not likely be detected in experiments based on the cross-linkingproperties of BS³; correspondingly, heparin cross-linked by BS³ to¹²⁵I-sFRP-1 or a number of other heparin-binding tracer proteins (FIG.5) was not observed. Although the details of their interaction have notbeen fully defined, the ability of sFRP-ΔCRD (SEQ ID NO: 8) to enhancethe activity of Wg in the Arm stabilization assay distinguished it fromanother heparin-binding protein (data not shown) and indicated that itsassociation with Wg has biological relevance.

The carboxyl-terminal deletion mutants that retained the CRD wereremarkable for their relatively weak association with Wg. Bafico et al.(Bafico et al., J. Biol. Chem. 274:16180–16187, 1999), reported that asFRP-1 truncation mutant retaining the CRD was able to coprecipitatewith Wnt-1 and Wnt-2. Conversely, the experiments described abovedemonstrate that of all the truncation mutants, sFRP-Δ2 (SEQ ID NO: 6)exhibited an intermediate capacity to interact with Wg. This impliesthat sFRP-Δ2 (SEQ ID NO: 6) shares a portion of a Wnt binding epitopewith sFRP-ΔCRD (SEQ ID NO: 8) or that it contains another binding siteinvolving the CRD that was perturbed in the D1 and D3 mutants.

Previous studies involving co-expression of sFRP and Wnt proteins in thesame cells indicated that sFRP family members can inhibit Wnt signaling.This was true in early Xenopus embryos because co-injection of mRNAencoding sFRP and Wnt molecules blocked Wnt-dependent duplication of thedorsal axis (Leyns et al., Cell 88:747–756, 1997; Wang et al., Cell88:757–766, 1997; Wang et al., J. Biol. Chem. 271:4468–4476, 1996; Finchet al., Proc. Natl. Acad. Sci. U.S.A. 94:6770–6775, 1997; and Xu et al.,Development 125:4767–4776, 1998), and in transfected cells in culturewhere stabilization of β-catenin was inhibited (Lin et al., Proc. Natl.Acad. Sci. U.S.A. 94:11196–11200, 1997 and Bafico et al., J. Biol. Chem.274:16180–16187, 1999). In these instances, high local concentrations ofthe proteins would have been likely, corresponding to the high end ofthe sFRP-1 dose-response experiment in the present report that alsoresulted in Wnt inhibition. Surprisingly, the work described herein isthe first to show that sFRP can enhance Wnt signaling under at lowconcentrations. Biphasic regulation by sFRP-1 provides a mechanism tofacilitate the position-dependent properties of Wnt signaling; cells inclose proximity to sources of sFRP-1 would be more refractory to Wnts,whereas cells at a greater distance would have their response to Wntspotentiated by a lower sFRP-1 concentration.

The molecular mechanism responsible for biphasic modulation of Wgsignaling by sFRP-1 is believed to be the presence of two distinctbinding sites for sFRP-1 on Wg that vary in their affinity; binding tothe high affinity site promotes Wnt signaling, whereas binding to thelow affinity site inhibits it. Alternatively, a higher affinityinteraction of Wg with the carboxyl-terminal domain of sFRP-1 promotessignaling by presenting a favorable Wg conformation to Fz, whereasadditional lower affinity binding via the CRD competes with Fz. Thismight involve a single sFRP-1 molecule binding to one Wg molecule, butit also could entail two sFRP-1 molecules interacting with one Wg. Thecross-linking data indicates that sFRP-1 and Wg interacts with both 1:1and 2:1 stoichiometry. Only a very small percentage of sFRP-1 tracer wasdetected as a homodimer in the cross-linking experiments, indicatingthat 2:1 stoichiometry probably is not due to binding of an sFRP-1homodimer to Wg. Alternative mechanisms also could account for abiphasic pattern of regulation. For instance, sFRP-1 /Wg might act as anagonist at low sFRP-1 concentrations, but at high concentrations sFRP-1could interact with Fz or another cell surface component and blocksignaling.

Use of soluble sFRP-1 derivatives in the ELISA competition model didenable the comparison of the relative affinities of sFRP-1/Wg. Theapparent affinity was in the range of 10–30 nM, rather close to the 9 nMaffinity recently calculated for the interaction of XWnt8 and mFz8(Hsieh et al., Proc. Natl. Acad. Sci. U.S.A. 96:3546–3551, 1999). Inaddition to these approximations, the Arm stabilization assays showedthat recombinant sFRP-1 elicited a biological response at a subnanomolarconcentration and activation was maximal at 15 nM. The higherconcentrations required for inhibition of Wnt signaling might occur inrestricted locations near the sites of sFRP-1 synthesis.

The results described herein establish that sFRP-1 can bind Wg andregulate Wnt signaling. It is believed that other members of the sFRPsubfamily have similar properties, although much work will be requiredto define the specific relationships that govern the interactions of themany Wnts, sFRPs, and Fzs. Recent reports suggest that sFRP-1 hasproapoptotic activity (Melkonyan et al., Proc. Natl. Acad Sci. U.S.A.94:13636–13641, 1997) and is up-regulated in certain settings followingserum withdrawal (Zhou et al., Int. J. Cancer 78:95–99, 1998). Itschromosomal locus at 8p11–12 (Finch et al., Proc. Natl. Acad. Sci.U.S.A. 94:6770–6775, 1997) is a site associated with loss ofheterozygosity for a variety of malignancies, and sFRP-1 expression isabsent from a high percentage of human breast tumor specimens (Ugoliniet al., Oncogene 18:1903–1910, 1999). Taken together, these observationsindicate that sFRP-1 and fragments thereof function as a tumorsuppressor, consistent with its ability to inhibit Wnt signaling at highconcentrations.

C. Methods

Cell Culture—MDCK cells (American Type Culture Collection) were grown inDulbecco's modified Eagle's medium (Life Technologies, Inc., Rockville,Md.) containing 10% fetal calf serum (Colorado Serum Company, Denver,Colo.) in 5% CO₂ at 37° C. Drosophila S2 cells and S2HSWg cellstransfected with a heat shock promoter/Wg construct (Bellahcene et al.,J Bone Miner Res 11(5):665–70, 1996 and Waltregny et al., J Natl. CancerInst. 90(13):1000–8, 1998), and S2 cells expressing DFz2 (Koeneman etal., Prostate 39(4):246–61, 1999) were kindly provided by the Nusse lab.All three S2 lines were cultured in Schneider's Drosophila medium (LifeTechnologies, Inc.) supplemented with 10% fetal calf serum, 100 units/mlpenicillin, and 100 mg/ml streptomycin at 25° C. in atmospheric air.Wg-containing and S2 control media were generated as describedpreviously (Ryden et al., Eur. J Biochem. 184(2):331–6, 1989).

Immunoblotting and Immunoprecipitation—Proteins resolved by SDS-PAGEwere transferred to Immobilon-P membranes (Millipore, Mass.). Unlessstated otherwise, all subsequent steps were performed at roomtemperature. After brief washing in phosphate-buffered saline (PBS),membranes were blocked with 3% nonfat dry milk in TTBS (20 mM Tris-HCl,pH 8.0, 0.05% Tween-20, 150 mM NaCl) for 2 h. Following five washes withTTBS, membranes were incubated for 2 h with primary antibodies diluted1:1000 (for a typical final concentration of 1–2 mg/ml) in 0.5% bovineserum albumin (BSA)/TTBS. sFRP-1 rabbit antisera were raised eitheragainst a synthetic amino-terminal peptide (Jarvis and Vedros, Infect.Immun. 55(1):174–80, 1987) or the full-length, purified protein.Monoclonal antibody to Wg, prepared by known techniques. Antibodies tothe c-Myc and polyhistidine epitopes were from Invitrogen (Carlsbad,Calif.). After five washes with TTBS, membranes were incubated for 1 hwith horseradish peroxidase conjugated to anti-mouse or anti-rabbitsecondary antibodies (Amersham Pharmacia Biotech, Uppsala, Sweden)diluted 1:2000 in 0.5% BSA/TTBS. Following five more washes with TTBS,bound anti-bodies were visualized by chemiluminescence (AmershamPharmacia Biotech) using X-Omat AR film (Kodak).

For immunoprecipitation, Wg-containing medium (80 ml) was preincubatedwith individual sFRP-1 derivatives (300 nM) for 10 min at roomtemperature. Subsequently, anti-Myc (0.2 mg) was added to the samples,which were then incubated overnight at 4° C. Sample volumes wereadjusted to 500 ml with lysis buffer (50 mM HEPES, pH 7.5, 50 mM NaCl,1% Triton X-100, 5 mM EDTA, 50 mM NaF, 6.7 mM Na4 P2 O7, 1 mM NaVO4, 10mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethyl-sulfonylfluoride) and 50 ml of a 50% protein G-Sepharose slurry (AmershamPharmacia Biotech) was added. After 1 h of incubation at 4° C. in arotary shaker, samples were washed three times with 1 ml of lysisbuffer. Final pellets were resuspended in 23×SDS sample buffer andboiled for 4 min, and the proteins were resolved by SDS-PAGE.

Expression, Purification, and Analysis of Recombinant sFRP-1 and ItsDerivatives—The human sFRP-1 NotI-SmaI cDNA fragment (33) was subclonedinto an XhoI site in the pcDNA3.1 expression vector (Invitrogen,Carlsbad, Calif.). To prepare derivatives containing c-Myc andpolyhistidine epitopes at their carboxyl termini, cDNAs encodingfull-length sFRP-1 or deletion mutants were generated by polymerasechain reaction with primers that introduced EcoRV and HindIIIrestriction sites at the 59 and 39 ends, respectively. The sequencescomprising the various derivatives are indicated in FIG. 2A. Purifiedpolymerase chain reaction products were ligated into the pcDNA3.1Myc/His, C(2) expression vector (Invitrogen), and plasmid samplesprepared from transformed DH5α competent cells (Life Technologies,Inc.). The fidelity of cDNAs was verified by sequence analysis.

MDCK cells (1.5×10⁶) were transfected with 10 mg of DNA of the varioussFRP-1 constructs, using the calcium phosphate precipitation method.Mass cultures were selected with Geneticin (500 mg/ml) for 21 days. Toisolate clonal cell lines, mass cultures were subcultured at a 1:50,000dilution in collagen-coated wells and subsequently transferred toculture dishes for further analysis. Expression of recombinant proteinwas determined by immunoblotting equal quantities of total protein fromconditioned medium and/or cell lysates. For large scale preparations,sFRP-1/MDCK transfectants were grown in T175 flasks until confluent.After washing with PBS, the cells were maintained in serum-freeDulbecco's modified Eagle's medium, and conditioned media were collectedevery 3 days for five to seven consecutive harvests. Media wereclarified by centrifugation at 10,000×g for 10 min at 4° C. andfiltration (pore size, 0.4 mm; Corning). Subsequently, media wereconcentrated 40-fold by ultrafiltration in a stirred chamber apparatus(Amicon M2000) using a Millipore YM membrane with either a 10- or 3-kDamolecular mass cut-off. Concentrated samples were snap-frozen forsubsequent purification.

Native sFRP-1 was purified with HiTrap-Heparin columns (AmershamPharmacia Biotech) equilibrated with PBS/0.3 M NaCl. After applying thesample to the column, the resin was washed with 10 column volumes ofequilibration buffer. Protein was eluted with a step gradient ofincreasing NaCl concentration. Aliquots from representative fractionswere resolved by SDS-PAGE and analyzed by immuno-blotting or silverstaining (Bio-Rad). sFRP-1 derivatives containing Myc/histidine epitopeswere purified in a similar manner, only using HiTrap Chelating Affinitycolumns (Amersham Pharmacia Biotech). The resin (1.0 ml) was washed with5.0 ml of water, charged with 0.5 ml of 0.1 M NiSO₄, and washed againwith 5.0 ml of water. Following equlibration with 50 mM phosphate/10 mMimidazole buffer (pH 7.4), protein was eluted with a step gradient ofincreasing imidazole concentration. Selected fractions were analyzed byimmunoblotting and silver staining. Typically, sFRP-1 derivatives wererecovered with 0.1 M imidazole. The identity of individual sFRP-1preparations was verified by microsequencing with an Applied Biosystems(Foster City, Calif.) protein sequencer (model 476). For sFRP-ΔCRD (SEQID NO: 8), 30 rounds of Edman degradation were carried out to ensurethat the entire CRD was deleted.

sFRP-1/Wg ELISA Binding Assays—sFRP-1 diluted in 0.02% NaN₃/PBS wasincubated in 96-well Falcon ELISA plates (300 ng/50 ml/well) for 2 h at37° C. After decanting, all wells were filled with 4% BSA/0.02% NaN₃/PBSand incubated for an additional 2 h at 37° C. Following five washes withTAPS (0.05% Tween-20 in 0.02% NaN₃/PBS), 50-ml aliquots of Wg-containingor S2 control medium diluted in 1% BSA/TAPS were incubated overnight atroom temperature. After five washes with TAPS, 50 ml/well of Wg mAbdiluted in 1% BSA/TAPS to a final concentration of 1 mg/ml was incubatedin wells for 2 h at 37° C. Another five washes in TAPS were followed bya 2 h treatment at 37° C. with 1:400 dilution of conjugated alkalinephosphatase-goat anti-mouse IgG (Sigma). After a final set of fivewashes with TAPS, 2 mg/ml p-nitrophenolphosphate (Sigma) in carbonatebuffer (0.1 M Na₂ CO₃, 1 mM MgCl₂, pH 9.8) was added. Absorbance at 405nm was measured with an ELISA plate reader (Bio-Rad, Hercules, Calif.).When the solid phase assay was performed with the various sFRP-1derivatives, wells were coated with 60 nM solutions of the respectivederivatives. ELISA competition experiments were performed as describedabove, except the indicated concentrations of sFRP-1 derivatives werepreincubated with Wg conditioned medium for 45 min at room temperatureprior to addition to wells that had been coated with native sFRP-1.

Covalent Cross-linking—sFRP-1 was iodinated as described previously(Kovats et al., Science 248(4952):220–3, 1990). Briefly, 10 mg of sFRP-1was reacted with 1 mCi of Na 125 I in the presence of 30 mg/mlchloramine T for 30–60 s. After addition of 80 mg/ml sodiummetabisulfite, the reaction mixture was applied to a heparin-Sepharosecolumn (bed volume, 0.3 ml) equilibrated in 0.1% BSA/PBS. Labeled sFRP-1was eluted with equilibration buffer containing 1.0 M NaCl and stored infrozen aliquots. Approximately 50 ml of Wg-containing or control mediumwas incubated with 1 mCi of ¹²⁵I-sFRP-1 for 40 min at room temperature.In some experiments, varying concentrations of heparin (12 kDa fromporcine intestine; Fisher, Madison, Wis.) or unlabeled sFRP-1 were alsopresent (see figures for details). After addition of 1 mMbis(sulfosuccinimidyl) suberate (BS³) cross-linking agent (Pierce,Rockford, Ill.), the incubation continued for 20 min. The reaction wasquenched with 20 mM glycine/1 mM Tris-HCl, and the mixture was incubatedwith Wg mAb (10 mg/ml) overnight at 4° C. After addition of 0.5 ml oflysis buffer and 50 ml of a 50% protein G-Sepharose slurry, samples wereincubated for 1 h at 4° C. Beads were pelleted by centrifugation at1000×g for 3 min at 4° C. and washed three times with 1 ml of lysisbuffer. The final pellets were resuspended in 2×SDS sample buffer,boiled for 4 min, and briefly microfuged to facilitate transfer. Proteinsamples were resolved in 8% polyacrylamide gels by SDS-PAGE. Afterfixation in 20% methanol/10% acetic acid for 45 min, the gel was driedand exposed to X-Omat AR film (Kodak) for autoradiography.

Armadillo Stabilization Assay—This assay was performed as describedpreviously (Ryden et al., Eur J Biochem 184(2):331–336, 1989). The blotswere probed with two primary antibodies, mouse monoclonal anti-Armantibody N27A at 1:50 and mouse monoclonal anti-HSP70 at 1:200,000 andone secondary antibody, goat anti-mouse IgG conjugated to horseradishperoxidase (Bio-Rad). Immunoreactive protein bands were visualized bytreating the blots with ECL reagents (Amersham Pharmacia Biotech) andthen exposing them to x-ray film. Equal loading of total protein wasconfirmed by inspection of the HSP70 protein band in each sample lane.

EXAMPLES Example 1

Expression and Purification of sFRP Fragments and Variants Thereof

sFRP fragments and variants thereof may be purified from the MDCK cells(1.5×10⁶; ATCC NO. CCL-34) that were transfected with sFRP encodingvectors as described above. sFRP fragments and variants thereof may alsobe purified from a tissue source using conventional biochemicaltechniques, or produced recombinantly in either prokaryotic oreukaryotic cells using methods well-known in the art (for example, thosedescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1989). The recombinant expression of sFRPfragments is described in (Uren et al., Journal of Biological Chem.275:4374–4382, 2000). Furthermore, the nucleic acid sequences encodingsFRP are available on GenBank, and include the cDNA sequence shown inSEQ. ID NO: 1.

Recombinant sFRP fragments and variants thereof may be convenientlyobtained using commercial systems designed for optimal expression andpurification of fusion proteins. Such fusion proteins typically includea protein tag that facilitates purification. Examples of such systemsinclude: the pMAL protein fusion and purification system (New EnglandBiolabs, Inc., Beverly, Mass.); the GST gene fusion system (AmershamPharmacia Biotech, Inc., Piscataway, N.J.); and the pTrcHis expressionvector system (Invitrogen, Carlsbad, Calif.). For example, the pMALexpression system utilizes a vector that adds a maltose binding proteinto the expressed protein. The fusion protein is expressed in E. coli.and the fusion protein is purified from a crude cell extract using anamylose column. If necessary, the maltose binding protein domain can becleaved from the fusion protein by treatment with a suitable protease,such as Factor Xa. The maltose binding fragment can then be removed fromthe preparation by passage over a second amylose column. Eukaryoticexpression systems may also be employed, including Pichia, tobacco andBaculovirus expression systems, such as those available commerciallyfrom Invitrogen.

For each of these systems, the entire sFRP protein may be produced byligating the open reading frame (ORF) of sFRP into the vector. To ensureeffective expression, the ORF must be operably linked to the vector,i.e., must be joined such that the reading frame of the ORF is alignedwith the reading frame of the protein tag. Where fragments of sFRP areto be expressed, an ORF encoding the desired fragment may be amplifiedby polymerase chain reaction (PCR) from the sFRP cDNA, cloned, purifiedand then ligated into the expression vector. Alternatively, theamplified fragment may be ligated directly into the expression vector.It may also be possible, depending on the availability of suitablerestriction sites in the sFRP cDNA to obtain the desired fragment byappropriate restriction endonuclease digestion, such that it can bedirectly cloned into the expression vector.

Purification of the expressed protein can be achieved either using thepurification regimen appropriate for the expression tag (if a commercialexpression/purification system is used), or conventional affinitychromatography using antibodies, preferably monoclonal antibodies, thatrecognize the appropriate regions of sFRP may be employed.

Where sFRP fragments are to be used, such fragments may alternatively begenerated through digestion of the full-length sFRP protein with variousproteases. The fragments may then be separated based on their uniquesize, charge or other characteristics. sFRP fragments may also besynthetically generated through the use of known peptide synthesismethods.

Example 2

Methods of Developing Screening Assays for Molecules that Modulate WntProtein Activity

In light of the present disclosure, one of ordinary skill in the art isenabled to practice new screening methodologies that are useful for theidentification of proteins and other compounds which bind to, orotherwise directly interact with, the complex formed by sFRP orfragments thereof and Wnt (sFRP/Wnt). The proteins and compounds includeendogenous cellular components which disrupt the binding of sFRP/Wnt.Thus, in one series of embodiments, cell lysates or tissue homogenatesmay be screened for proteins or other compounds that disrupt sFRP/Wntbinding. Alternatively, any of a variety of exogenous compounds, bothnaturally occurring and/or synthetic (e.g., libraries of small moleculesor peptides), may be screened for the ability to disrupt sFRP/Wnt. Smallmolecules are particularly preferred in this context because they aremore readily absorbed after oral administration, have fewer potentialantigenic determinants, and/or are more likely to cross the blood brainbarrier than larger molecules such as nucleic acids or proteins.

Furthermore, the identification of deletion mutants (i.e., sFRP-ΔCRD,SEQ ID NO: 8) that are significantly smaller than full length sFRP butyet maintain the ability to bind to and regulate Wnt activity can serveas “lead compounds” in the design and development of newpharmaceuticals. For example, as is well known in the art, sequentialmodification of small molecules (e.g., amino acid residue replacementwith peptides; functional group replacement with peptide or non-peptidecompounds) is a standard approach in the pharmaceutical industry for thedevelopment of new pharmaceuticals. Such development generally proceedsfrom a “lead compound” which is shown to have at least some of theactivity (e.g., low concentrations of sFRP-ΔCRD (SEQ ID NO: 8) increasesWnt activity) of the desired pharmaceutical. In particular, when one ormore compounds having at least some activity of interest (e.g.,modulation of Wnt activity) are identified, structural comparison of themolecules can greatly inform the skilled practitioner by suggestingportions of the lead compounds which should be conserved and portionswhich may be varied in the design of new candidate compounds. Thus, thepresent invention also provides potential lead compounds as well asmeans of identifying such lead compounds which may be sequentiallymodified to produce new candidate compounds for use in the treatment ofdiseases associated with abnormal Wnt activity, i.e., cancer. These newcompounds then may be tested both for Wnt-binding or blocking (e.g., inthe binding assays described above) and for biological efficacy (e.g.,in the Arm assay described herein). This procedure may be iterated untilcompounds having the desired therapeutic activity and/or efficacy areidentified.

The effect of agents that disrupt sFRP/Wnt binding can be monitoredusing the Arm assay described above. Agents that disrupt sFRP/Wntbinding and enhance Wnt signaling are useful for treating conditionsassociated with decreased Wnt activity and agents that are found todisrupt sFRP/Wnt binding and increase Wnt activity are useful fortreating diseases associated with increased Wnt activity such asneoplasia development. Methods of detecting such binding include thecross-linking assay described above as well as other methods thatinvolve monitoring changes in fluorescence, molecular weight, or theconcentration of either Wnt or sFRP, either in a soluble phase or in asubstrate-bound phase.

Once identified by the methods described above, the candidate compoundsmay then be produced in quantities sufficient for pharmaceuticaladministration or testing (e.g., .mu.g or mg or greater quantities), andformulated in a pharmaceutically acceptable carrier (see, e.g.,Remington's Pharmaceutical Sciences, Gennaro, A., ed., Mack Pub., 1990).These candidate compounds may then be administered to the transformedcells of the invention, to the transgenic animal models of theinvention, to cell lines derived from the animal models or from humanpatients.

The proteins or other compounds identified by these methods may bepurified and characterized by any of the standard methods known in theart. Proteins may, for example, be purified and separated usingelectrophoretic (e.g., SDS-PAGE, 2D PAGE) or chromatographic (e.g.,HPLC) techniques and may then be microsequenced. For proteins with ablocked N-terminus, cleavage (e.g., by CNBr and/or trypsin) of theparticular binding protein is used to release peptide fragments. Furtherpurification/characterization by HPLC and microsequencing and/or massspectrometry by conventional methods provides internal sequence data onsuch blocked proteins. For non-protein compounds, standard organicchemical analysis techniques (e.g., IR, NMR and mass spectrometry;functional group analysis; X-ray crystallography) may be employed todetermine their structure and identity.

Methods for screening cellular lysates, tissue homogenates, or smallmolecule libraries for candidate sFRP/Wnt disrupting molecules are wellknown in the art and, in light of the present disclosure, may now beemployed to identify compounds which disrupt such binding and increaseor decrease Wnt biological activity.

In light of the present disclosure, a variety of affinity bindingtechniques well known in the art may be employed to isolate proteins(i.e. lead compounds) or other compounds which disrupt sFRP/Wnt binding.In general, sFRP or a fragment thereof can be immobilized on a substrate(e.g., a column or filter) and a solution containing Wnt protein can beintroduced to the column to allow formation of the sFRP/Wnt complex.Then a solution including the test compound(s) is introduced to thecolumn under conditions which are permissive for binding. The substrateis then washed with a solution to remove unbound or weakly boundmolecules. A second wash may then elute those compounds which stronglybound to the immobilized sFRP. Alternatively, the test compounds may beimmobilized and a solution containing sFRP/Wnt may be contacted with thecolumn, filter or other substrate. The ability of either the sFRP orfragment thereof, or the Wnt protein to bind to the test compound may bedetermined as above.

In other embodiments the invention provides for methods of identifyingcompounds with the ability to modulate the activity of Wnt proteins.Furthermore, the identification of the biphasic nature of sFRP/Wntinteractions allows for the development of compounds that canspecifically modulate increases and decreases in Wnt biologicalactivity. Using the Arm assay described above modifications in the sFRPprotein and fragments thereof can be sequentially made. These modifiedproteins can then be tested for their ability to increase Wnt activity.In other words the discovery that sFRP and fragments thereof can causean increase of Wnt activity can be exploited to identify sFRP mutantswith enhance Wnt inducing activity.

Example 3

Assessing sFRP and Fragments Thereof for Their Ability to Modulate WntBiological Activity

Following the purification of sFRP or a fragment of sFRP, the biologicalactivity can be assessed using the methods described above.Specifically, the Arm assay can be used to determine the ability of thesFRP fragment or variant thereof to modulate Wnt activity. Similarly, anassay for β-catenin showing biochemical response to Wnts can be alsoused to monitor Wnt biological activity (Papkoff et al., Mol. Cell Biol.16:2128–2134, 1996; and Shimizu et al. Cell Growth and Diff.8:1349–1358, 1997, which are herein incorporated by reference). Finally,the ability of sFRP binding fragments and mimetics thereof to bind toWnt and modulate its activity can be tested using the cross-linkingassays described above.

Example 4

Sequence Variants

While the amino acid sequence of the prototypical human sFRP protein isprovided in SEQ. ID NO: 3, and the sequence of a cDNA molecule encodingthis protein is given in SEQ. ID NO: 2, one of skill in the art willappreciate that the practice of this invention is not limited to theseprecise sequences. Thus, the invention may be practiced with moleculesthat differ from the exact molecules disclosed, but which retain therequisite biological activity.

Furthermore, variants of sFRP fragments that have been modified suchthat they bind to Wnt proteins but do not contain the CRD region ofparticular interest. These variants will retain the ability tospecifically bind Wnt proteins.

As mentioned above, the fragments and variants of sFRP described supra,are characterized by their ability to modulate Wnt biological activity.This ability, however, is concentration dependant and a lowconcentration of sFRP fragments may serve to enhance Wnt activity and athigh concentrations it may serve to suppress Wnt activity. When sFRPfragments and variants thereof are used to suppress Wnt activity, theycan be used to modulate conditions associated with increased Wntactivity such as tumor growth. When used to inhibit tumor growth a givensFRP fragment, such as the sFRP-1-ΔCRD fragment (SEQ ID NO: 8), will befound to be biologically active if it causes at least 30% inhibition oftumor growth when compared to a non-treated control. However, it islikely that some therapeutically active fragments and variants of sFRPwill show an increased level of Wnt inhibition. For example, somevariants and fragments of sFRP will show at least 40% inhibition, atleast 50% inhibition, at least 60% inhibition, or at least 70%inhibition. Similarly, the biphasic nature of sFRP and fragments thereofmeans that these polypeptides can be used to increase Wnt activity.Moreover, using the ARM assays described above it is now possible toindividually assess the biological activity of a given variant orfragment of sFRP, and to determine if it can cause and increase or adecrease in Wnt activity.

The therapeutically effective fragments and variants of sFRP are alsocharacterized by the number of amino acid residues that they contain.For example, in some instances it may be desirable to use relativelyshort fragments and variants of sFRP. These short fragments and variantsof sFRP may contain at least 5, 10, 20, or 30 contiguous amino acidsresidues of the sFRP sequence. However, such short fragments andvariants of sFRP will maintain the ability to bind Wnt proteins.

Additionally, it is possible to vary the cDNA sequences encodingtherapeutically effective fragments or variants of sFRP while stillencoding a protein having the desired biological activity. In theirsimplest form, such sequence variants may differ from the disclosedsequences by alteration of the coding region to fit the codon usage biasof the particular organism into which the molecule is to be introduced.Additionally, the coding region may be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence in sucha way that, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequence identicalor substantially similar to the disclosed sFRP protein sequence. Forexample, the seventh amino acid residue of the sFRP-1-ΔCRD fragment (SEQID NO: 8) is Glu, (E). This is encoded in the sFRP-1-ΔCRD open readingframe (ORF) by the nucleotide codon triplet GAG. Because of thedegeneracy of the genetic code, one other nucleotide codon, GAA, alsoencodes for glutamic acid. Thus, the nucleotide sequence of thesFRP-1-ΔCRD ORF could be changed at this position to GAA withoutaffecting the amino acid composition of the encoded protein or thecharacteristics of the protein.

As previously mentioned, the invention may also be practiced with sFRPfragments that vary in amino acid sequence from the sequence shown inSEQ. ID NO: 2. These variants include proteins that differ in amino acidsequence from the disclosed sequence but which retain the ability tobind Wnt proteins. Such proteins may be produced by manipulating thenucleotide sequence of ORF that encodes the protein, for example bysite-directed mutagenesis or the polymerase chain reaction. The simplestmodifications involve the substitution of one or more amino acids foramino acids having similar biochemical properties. These so-calledconservative substitutions are likely to have minimal impact on theactivity of the resultant protein.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Such substitutionsgenerally are conservative when it is desired to finely modulate thecharacteristics of the protein. Examples of amino acids which may besubstituted for an original amino acid in a protein and which areregarded as conservative substitutions include: Ser for Ala; Lys forArg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln; Asp forGlu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; lie or Val forLeu; Arg or Gln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe;Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile orLeu for Val.

More substantial changes in function or other features may be obtainedby selecting substitutions that are less conservative than thosedescribed above, i.e., selecting residues that differ more significantlyin their effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. The substitutionswhich in general are expected to produce the greatest changes in proteinproperties will be those in which (a) a hydrophilic residue, e.g., serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histadyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine. The effects of these amino acid substitutions ordeletions or additions may be assessed through the use of the biologicalassays described above.

Example 5

Use of sFRP-1 to Increase Wnt Activity

Transgenic mice that have had specific Wnt genes deleted (knockout mice)display specific developmental disorders, such as Wnt-4 knockout micewhich fail to develop kidneys and female organs, and Wnt-7a knockoutmice which display defects in limb development (Stark et al., Nature372:679–683, 1994; Vainio et al., Nature 397:405–409, 1999; Parr et al.,Nature 374:350–353, 1995). Consistent with the above references Wntexpression has been observed to fluctuate during the estrous cycle(Miller et al., Mech. Of Development 76:91–99, 1998). Hence, sFRP andfragments thereof are believed to be useful for increasing Wnt activityin conditions that are characterized by developmental disorders, such asrenal agenesis.

Example 6

Incorporation of Therapeutically Effective Fragments and Variants ofsFRP into Pharmaceutical Compositions

For administration to animals, purified sFRP fragments or variantsthereof are generally combined with a pharmaceutically acceptablecarrier. Pharmaceutical preparations may contain only a single sFRPfragment, or may be composed of more than one variety of sFRP fragments.In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol, human albumin orthe like as a vehicle. For solid compositions (e.g., powder, pill,tablet, or capsule forms), conventional non-toxic solid carriers caninclude, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

As is known in the art, protein-based pharmaceuticals may be onlyinefficiently delivered through ingestion. However, pill-based forms ofpharmaceutical proteins may be alternatively be administeredsubcutaneously, particularly if formulated in a slow-releasecomposition. Slow-release formulations may be produced by combining thetarget protein with a biocompatible matrix, such as cholesterol. Anotherpossible method of administering protein pharmaceuticals is through theuse of mini osmotic pumps. As stated above a biocompatible carrier wouldalso be used in conjunction with this method of delivery.

It is also contemplated that sFRP fragments could be delivered to cellsin the nucleic acid form and subsequently translated by the host cell.This could be done, for example through the use of viral vectors orliposomes. Liposomes could also be used for the delivery of the proteinitself.

The pharmaceutical compositions of the present invention may beadministered by any means that achieve their intended purpose. Amountsand regimens for the administration of sFRP fragments can be determinedreadily by those with ordinary skill in the clinical art of treatingconditions associated with abnormal Wnt activity. For use in treatingthese conditions, the described proteins are administered in an amounteffective to either increase Wnt biological activity or decrease Wntbiological activity. Doses sufficient to achieve a tissue concentrationthat causes an increase or a decrease in Wnt biological activity may bedetermined by using the amounts described in the forgoing examples. Thepeptides or proteins may be administered to a host in vivo, such as forexample, through systemic administration, such as intravenous orintraperitoneal administration. Also, the peptides or proteins may beadministered intralesionally: i.e. the peptide or protein is injecteddirectly into the tumor or affected area.

Effective doses of sFRP fragments for therapeutic application will varydepending on the nature and severity of the condition to be treated, theage and condition of the subject and other clinical factors. Thus, thefinal determination of the appropriate treatment regimen will be made bythe attending clinician. Typically, the dose range will be from about0.1 μg/kg body weight to about 100 mg/kg body weight. Other suitableranges include doses of from about 1 μg/kg to 10 mg/kg body weight. Thedosing schedule may vary from once a week to daily depending on a numberof clinical factors, such as the subject's sensitivity to the protein.Examples of dosing schedules are 3 μg/kg administered twice a week,three times a week or daily; a dose of 7 μg/kg twice a week, three timesa week or daily; a dose of 10 μg/kg twice a week, three times a week ordaily; or a dose of 30 μg/kg twice a week, three times a week or daily.In the case of a more aggressive disease it may be preferable toadminister doses such as those described above by alternate routesincluding intravenously or intrathecally. Continuous infusion may alsobe appropriate.

Having illustrated and described the principles of the invention inmultiple embodiments and examples, it should be apparent to thoseskilled in the art that the invention can be modified in arrangement anddetail without departing from such principles. We claim allmodifications coming within the spirit and scope of the followingclaims.

1. An isolated polynucleotide that encodes a polypeptide fragment ofsecreted frizzled related protein (sFRP) comprising an amino acidsequence at least 90% homologous to the amino acid sequence as set forthin SEQ ID NO: 8, wherein the encoded polypeptide fragment binds towingless protein.
 2. The isolated polynucleotide according to claim 1wherein the polynucleotide comprises the nucleic acid sequence shown inSEQ ID. NO:
 13. 3. A vector comprising the nucleic acid sequence ofclaim
 1. 4. An isolated host cell comprising the vector of claim
 3. 5. Amethod of producing a polypeptide fragment of sFRP comprising the stepsof: (a) providing a host cell according to claim 4; (b) culturing saidhost cell under conditions such that the polypeptide fragment of sFRP isproduced by said host cell; and (c) isolating said polypeptide fragmentof sFRP.
 6. The isolated polynucleotide of claim 1, wherein thepolynucleotide encodes a polypeptide fragment of secreted frizzledrelated protein (sFRP) comprising an amino acid sequence at least 95%homologous to the amino acid sequence as set forth in SEQ ID NO: 8,wherein the encoded polypeptide fragment binds to wingless protein. 7.The isolated polynucleotide of claim 1, wherein the polynucleotideencodes a polypeptide fragment of secreted frizzled related protein(sFRP) comprising an amino acid sequence at least 98% homologous to theamino acid sequence as set forth in SEQ ID NO: 8, wherein the encodedpolypeptide fragment binds to wingless protein.
 8. The isolatedpolynucleotide of claim 1, wherein the polynucleotide encodes apolypeptide fragment of secreted frizzled related protein (sFRP)comprising the amino acid sequence as set forth in SEQ ID NO: 8, whereinthe encoded polypeptide fragment binds to wingless protein.
 9. A vectorcomprising the isolated nucleic acid of claim
 2. 10. A vector comprisingthe isolated nucleic acid of claim
 6. 11. A vector comprising theisolated nucleic acid of claim
 7. 12. A vector comprising the isolatednucleic acid of claim
 8. 13. An isolated host cell comprising the vectorof claim
 9. 14. An isolated host cell comprising the vector of claim 10.15. An isolated host cell comprising the vector of claim
 11. 16. Anisolated host cell comprising the vector of claim
 12. 17. A method ofproducing a polypeptide fragment of sFRP comprising the steps of: (a)providing a host cell according to claim 13; (b) culturing the host cellunder conditions such that the polypeptide fragment of sFRP is producedby said host cell; and (c) isolating said polypeptide fragment of sFRP.18. A method of producing a polypeptide fragment of sFRP comprising thesteps of: (a) providing a host cell according to claim 14; (b) culturingsaid host cell under conditions such that the polypeptide fragment ofsFRP is produced by said host cell; and (c) isolating said polypeptidefragment of sFRP.
 19. A method of producing a polypeptide fragment ofsFRP comprising the steps of: (a) providing a host cell according toclaim 15; (b) culturing said host cell under conditions such that thepolypeptide fragment of sFRP is produced by said host cell; and (c)isolating said polypeptide fragment of sFRP.
 20. A method of producing apolypeptide fragment of sFRP comprising the steps of: (a) providing ahost cell according to claim 16; (b) culturing said host cell underconditions such that the polypeptide fragment of sFRP is produced bysaid host cell; and (c) isolating said polypeptide fragment of sFRP.